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Team:Newcastle/31 August 2010
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==Aim== | ==Aim== | ||
| - | The aim of | + | The aim of this experiment is to amplify plasmid linearized pSB1C3 for the construction of [[Team:Newcastle/Urease|''rocF'' BioBrick]] with the help of a single Phusion PCR using old primers. |
==Materials and Protocol== | ==Materials and Protocol== | ||
Revision as of 09:47, 31 August 2010
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Contents |
Amplification of the plasmid PSB1C3 for rocF BioBrick
Aim
The aim of this experiment is to amplify plasmid linearized pSB1C3 for the construction of rocF BioBrick with the help of a single Phusion PCR using old primers.
Materials and Protocol
Please refer to PCR for Phusion PCR protocol. The details for the 6 PCR reactions are mentioned below:
PCR
| Tube | Part to be amplified | DNA fragment consisting the part | Forward primer | Reverse Primer | Melting Temperature (Tm in °C) | Size of the fragment (in bp) | Extension time* (in seconds) |
|---|---|---|---|---|---|---|---|
| 1 | Plasmid Vector | pSB1C3 | P1V1 forward | P2V1 reverse | 58 | 2072 approx. | 60 |
| 2 | Pspacoid Promoter | pMutin4 | P1P1 forward | P2P1 reverse | 49 | 106 approx. | 15 |
| 3 | 1st fragment of rocF CDS | B. subtilis 168 chromosome | P1S1 forward | P2S1 reverse | 58 | 246 approx. | 15 |
| 4 | 2nd fragment of rocF CDS | B. subtilis 168 chromosome | P3S2 forward | P4S2 reverse | 65 | 597 approx. | 20 |
| 5 | 3rd fragment of rocF CDS | B. subtilis 168 chromosome | P5S3 forward | P6S3 reverse | 66 | 125 approx. | 15 |
| 6 | Double Terminator | pSB1AK3 consisting BBa_B0014 biobrick | P1T1 forward | P2T1 reverse | 56 | 116 approx. | 15 |
Table 1: Table represents 6 different Phusion PCR reactions and the parts which are amplified so that they can be ligated together with the help of Gibson Cloning method.
- The extension rate of the Phusion polymerase is 1Kb/ 30 seconds. Thus the extension time of each and every PCR reaction is slightly different.
- For learning about the rocF fragments, please refer to the Cloning strategy for rocF.
Discussion
All the 6 Phusion PCR reactions were done however, gel electrophoresis was not done today to check whether the fragments have actually amplified or not.
Conclusion
Tomorrow 5th August, 2010, we would be running gel electrophoresis to check the outcome of the 6 PCR reactions and later all the fragments will be ligated with help of Gibson protocol.
   
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